Obesity and diabetes are related public health issues that have become increasingly prominent in the past few decades. Obese individuals have an increased risk for developing Type II diabetes suggesting shared pathophysiology. In fact, obese individuals develop diabetes when they develop insulin resistance, in which tissues no longer respond appropriately to insulin. In particular, adipose tissue is an important site of insulin action, thus resistance in this tissue has major repercussions for whole body energy homeostasis. During fasting, adipocytes, cells of adipose tissue, break down their triglyceride stores (lipolysis) to provide energy for other tissues. Conversely, glucose uptake and suppression of lipolysis is mediated by insulin signaling. However, the mechanism by which insulin opposes lipolysis is still not fully understood. In preliminary studies, we have shown this regulation to be highly localized to lipid droplets, the site of triglyceride storage in fat cells. In this proposal, I set out to elucidate signaling complexes formed by key scaffolding proteins, caveolins and A kinase anchoring proteins (AKAPs) at lipid droplets that organize the spatiotemporal regulation of lipolysis upon stimulation with catecholamine (lipolytic) and insulin (anti- lipolytic). Specifically, I will isolate lipid droplets from 3T3L1 adipocytes via density gradient centrifugation method and probe the fractions for specific isoforms of caveolins and AKAPs upon lipolytic and anti-lipolytic stimuli in order to characterize the conditions under which these proteins dynamically associate with lipid droplets. Furthermore, I will identify the proteins that associate with these scaffolders via co-immunoprecipitation experiments and immunofluorescence staining. Finally, I set out to determine the functional role of these complexes at lipid droplets in the context of lipolysis by measuring glycerol release and PKA activity upon genetic knockdown of caveolin and AKAPs using RNAi approach, as well as upon inhibition and displacement of the components in the complexes. For example, I will generate a dominant negative form of PDE3B that will compete with the endogenous PDE3B for binding to caveolin-1, thus dissociating the protein from signaling complexes at the lipid droplet without effecting the overall PDE activity within the cell. While important information can be collected from these proposed experiments, they only provide a snapshot of what is going on in the cell. To achieve a higher level of spatial and temporal resolution, I will use FRET-based biosensors of cAMP and PKA activity targeted to lipid droplets to monitor the effect of such perturbations on cAMP/PKA signaling in real time using fluorescence microscopy. Ultimately, I aim to address existing gaps in our understanding of the molecular mechanisms that are responsible for the dynamic regulation of lipolysis in hopes to provide key insights into the defects in insulin resistant tissue that contribute to disease progression. The information generated by this study will aid in the development of new therapeutics as well as our understanding of the mechanisms behind the spatiotemporal regulation of cellular signaling. PUBLIC HEALTH RELEVANCE: To ensure normal energy balance in the body, opposing signaling pathways regulate the breakdown of triglycerides in adipose tissue: catecholamines stimulate and insulin inhibits this process. However in diabetic and many obese individuals, fat and other tissues become unresponsive to insulin leading to high levels of glucose and fatty acids in the blood. Thus, understanding how insulin regulates the breakdown of fat in normal functioning adipose tissue will provide important insights into the defects of resistant tissue and lead to the development of new therapeutics.